Flavivirus vaccines

ABSTRACT

The invention provides flavivirus vaccines and methods of making and using these vaccines.

This application is a continuation of U.S. Ser. No. 13/668,819, filedNov. 5, 2012 (U.S. Pat. No. 8,852,914), which is is a continuation ofU.S. Ser. No. 13/423,746, filed Mar. 19, 2012 (abandoned), which is acontinuation of U.S. Ser. No. 13/198,976, filed Aug. 5, 2011(abandoned), which is a continuation of U.S. Ser. No. 12/962,216, filedDec. 7, 2010, (abandoned) which is a continuation of U.S. Ser. No.12/325,864, filed Dec. 1, 2008 (abandoned), which is a continuation ofU.S. Ser. No. 10/345,036, filed Jan. 15, 2003 (U.S. Pat. No. 7,459,160),which claims priority from U.S. Provisional patent application Ser. Nos.60/348,949, filed Jan. 15, 2002, and 60/385,281, filed May 31, 2002, thecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to flavivirus vaccines.

BACKGROUND OF THE INVENTION

Flaviviruses are small, enveloped, positive-strand RNA viruses, severalof which pose current or potential threats to global public health.Yellow fever virus, for example, has been the cause of epidemics incertain jungle locations of sub-Saharan Africa, as well as in some partsof South America. Although many yellow fever infections are mild, thedisease can also cause severe, life-threatening illness. The diseasestate has two phases. The initial or acute phase is normallycharacterized by high fever, chills, headache, backache, muscle aches,loss of appetite, nausea, and vomiting. After three to four days, thesesymptoms disappear. In some patients, symptoms then reappear, as thedisease enters its so-called toxic phase. During this phase, high feverreappears and can lead to shock, bleeding (e.g., bleeding from themouth, nose, eyes, and/or stomach), kidney failure, and liver failure.Indeed, liver failure causes jaundice, which is yellowing of the skinand the whites of the eyes, and thus gives “yellow fever” its name.About half of the patients who enter the toxic phase die within 10 to 14days. However, persons that recover from yellow fever have lifelongimmunity against reinfection. The number of people infected with yellowfever virus over the last two decades has been increasing, with therenow being about 200,000 yellow fever cases, with about 30,000 deaths,each year. The re-emergence of yellow fever virus thus presents aserious public health concern.

Dengue (DEN) virus is another example of a flavivirus. Dengue virusesare transmitted to humans by mosquitoes (mainly by Aedes aegypti) andare the cause of a growing public health problem worldwide. Fifty to onehundred million persons are infected by Dengue virus annually, and ratesof infection as high as 6% have been observed in some areas (Gubler,“Dengue and Dengue Hemorrhagic Fever,” CABI Publ., New York, Chapter 1,pp. 1-22, 1997; Burke et al., Am. J. Trop. Med. Hyg. 38:172-180, 1988).Four serotypes of Dengue virus (dengue types 1-4) circulate in theCaribbean, Asia, and the Americas. The severe, potentially lethal formof DEN infection [dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS)] is an immunopathological disease occurring in individuals whohave sustained sequential infections with different DEN serotypes. Over3.6 million cases of DHF and 58,000 deaths caused by DHF were reportedbetween 1980 and 1995 (Halstead, “Dengue and Dengue Hemorrhagic Fever,”CABI Publ., New York, Chapter 2, pp. 23-44, 1997). Because of thepathogenesis of DHF/DSS, it is generally thought that a optimal denguevaccine may need to immunize against all four serotypes of Dengue virussimultaneously and induce long-lasting immunity. Despite the extensiveefforts that have made towards developing an effective Dengue vaccinesince World War II, there is currently no approved dengue vaccineavailable.

Flaviviruses, including yellow fever virus and dengue virus, have twoprincipal biological properties responsible for their induction ofdisease states in humans and animals. The first of these two propertiesis neurotropism, which is the propensity of the virus to invade andinfect nervous tissue of the host. Neurotropic flavivirus infection canresult in inflammation and injury of the brain and spinal cord (i.e.,encephalitis), impaired consciousness, paralysis, and convulsions. Thesecond biological property of flaviviruses is viscerotropism, which isthe propensity of the virus to invade and infect vital visceral organs,including the liver, kidney, and heart. Viscerotropic flavivirusinfection can result in inflammation and injury of the liver(hepatitis), kidney (nephritis), and cardiac muscle (myocarditis),leading to failure or dysfunction of these organs. Neurotropism andviscerotropism appear to be distinct and separate properties offlaviviruses.

Some flaviviruses are primarily neurotropic (such as West Nile virus),others are primarily viscerotropic (e.g., yellow fever virus and denguevirus), and still others exhibit both properties (such as KyasanurForest disease virus). However, both neurotropism and viscerotropism arepresent to some degree in all flaviviruses. Within the host, aninteraction between viscerotropism and neurotropism is likely to occur,because infection of viscera occurs before invasion of the centralnervous system. Thus, neurotropism depends on the ability of the virusto replicate in extraneural organs (viscera). This extraneuralreplication produces viremia, which in turn is responsible for invasionof the brain and spinal cord.

One approach to developing vaccines against flaviviruses is to modifytheir virulence properties, so that the vaccine virus has lost itsneurotropism and viscerotropism for humans or animals. In the case ofyellow fever virus, two vaccines (yellow fever 17D and the Frenchneurotropic vaccine) have been developed (Monath, “Yellow Fever,” InPlotkin and Orenstein, Vaccines, 3^(rd) ed., 1999, Saunders,Philadelphia, pp. 815-879). The yellow fever 17D vaccine was developedby serial passage in chicken embryo tissue, and resulted in a virus withsignificantly reduced neurotropism and viscerotropism. The Frenchneurotropic vaccine was developed by serial passages in mouse braintissue, and resulted in loss of viscerotropism, but retainedneurotropism. A high incidence of neurological accidents (post-vaccinalencephalitis) was associated with the use of the French vaccine.Approved vaccines are not currently available for many medicallyimportant flaviviruses having viscerotropic properties, such as dengue,West Nile, and Omsk hemorrhagic fever viruses, among others.

Fully processed, mature virions of flaviviruses contain three structuralproteins, capsid (C), membrane (M), and envelope (E), and sevennon-structural proteins. Immature flavivirions found in infected cellscontain pre-membrane (prM) protein, which is a precursor to the Mprotein. The flavivirus proteins are produced by translation of asingle, long open reading frame to generate a polyprotein, followed by acomplex series of post-translational proteolytic cleavages of thepolyprotein, to generate mature viral proteins (Amberg et al., J. Virol.73:8083-8094, 1999; Rice, “Flaviviridae,” In Virology, Fields (ed.),Raven-Lippincott, New York, 1995, Volume I, p. 937). The virusstructural proteins are arranged in the polyprotein in the orderC-prM-E.

SUMMARY OF THE INVENTION

The invention provides flaviviruses including one or more hinge regionmutations that reduce viscerotropism of the flaviviruses. Theseflaviviruses can be, for example, yellow fever virus (e.g., a yellowfever virus vaccine strain); a viscerotropic flavivirus selected fromthe group consisting of Dengue virus, West Nile virus, Wesselsbronvirus, Kyasanur Forest Disease virus, and Omsk Hemorrhagic fever virus;or a chimeric flavivirus. In one example of a chimeric flavivirus, thechimera includes the capsid and non-structural proteins of a firstflavivirus virus (e.g., a yellow fever virus) and the pre-membrane andenvelope proteins of a second flavivirus (e.g., a Japanese encephalitisvirus or a Dengue virus (e.g., Dengue virus 1, 2, 3, or 4)) including anenvelope protein mutation that decreases viscerotropism of the chimericflavivirus. In the case of Dengue virus, the mutation can be, forexample, in the lysine at Dengue envelope amino acid position 202 or204. This amino acid can be substituted by, for example, arginine.

The invention also provides vaccine compositions that include any of theviruses described herein and a pharmaceutically acceptable carrier ordiluent, as well as methods of inducing an immune response to aflavivirus in a patient by administration of such a vaccine compositionto the patient. Patients treated using these methods may not have, butbe at risk of developing, the flavivirus infection, or may have theflavivirus infection.

Also included in the invention are methods of producing flavivirusvaccines, involving introducing into a flavivirus a mutation thatresults in decreased viscerotropism. Further, the invention includesmethods of identifying flavivirus (e.g., yellow fever virus or chimericflavivirus) vaccine candidates, involving (i) introducing a mutationinto the hinge region of a flavivirus; and (ii) determining whether theflavivirus including the hinge region mutation has decreasedviscerotropism, as compared with a flavivirus virus lacking themutation.

The flaviviruses of the invention are advantageous because, in havingdecreased viscerotropism, they provide an additional level of safety, ascompared to their non-mutated counterparts, when administered topatients. Additional advantages of these viruses are provided by thefact that they can include sequences of yellow fever virus strainYF17D(e.g., sequences encoding capsid and non-structural proteins), which (i)has had its safety established for >60 years, during which over 350million doses have been administered to humans, (ii) induces a longduration of immunity after a single dose, and (iii) induces immunityrapidly, within a few days of inoculation. In addition, the vaccineviruses of the invention cause an active infection in the treatedpatients. As the cytokine milieu and innate immune response of immunizedindividuals are similar to those in natural infection, the antigenicmass expands in the host, properly folded conformational epitopes areprocessed efficiently, the adaptive immune response is robust, andmemory is established. Moreover, in certain chimeras of the invention,the prM and E proteins derived from the target virus contain thecritical antigens for protective humoral and cellular immunity.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plaque size variants produced by ChimeriVax™-JE FRhL₃(large plaque, Panel A) and FRhL₅ (small plaque, Panel B). Plaques werestained using rabbit anti-JE antiserum followed by anti-rabbitIgG-horseradish peroxidase.

FIG. 2 is a series of graphs showing survival distributions of YF-VAX®and ChimeriVax™-JE constructs, with and without a mutation at E279(M→K). Four day-old suckling mice inoculated by the intracerebral routewith (FIG. 2A) approximately 0.7 log₁₀ PFU; (FIG. 2B) approximately 1.7log₁₀ PFU; and (FIG. 2C) ˜2.7 log₁₀ PFU.

FIG. 3 is a graph of regression analysis, mortality vs. virus dose,showing similar slopes and parallel lines for viruses with (FRhL₅) andwithout (FRhL₃) the Met to Lys reversion, allowing statisticalcomparison. The FRhL₅ virus was 18.52 times more potent (virulent) thanFRhL₃ (p<0.0001).

FIG. 4 shows the results of independent RNA transfection and passageseries of ChimeriVax™-JE virus in FRhL and Vero cells. The emergence ofmutations in the prME genes by passage level is shown.

FIG. 5 is a three-dimensional model of the flavivirus envelopeglycoprotein ectodomain showing locations of mutations in the hingeregion occurring with adaptation in FRhL or Vero cells. The sequence ofthe JE envelope glycoprotein (strain JaOArS982; Sumiyoshi et al.,Virology 161:497-510, 1987) was aligned to one of the TBE structuraltemplates (Rey et al., Nature 375:291-298, 1995) as an input forautomated homology modeling building by the method of SegMod (SegmentMatch Modeling) using LOOK software (Molecular Application Group, PaloAlto, Calif.).

DETAILED DESCRIPTION

The invention provides flaviviruses (e.g., yellow fever viruses andchimeric flaviviruses) having one or more mutations that result indecreased viscerotropism, methods for making such flaviviruses, andmethods for using these flaviviruses to prevent or to treat flavivirusinfection. The mutation (or mutations) in the flaviviruses of theinvention is present in the hinge region of the envelope protein, whichwe have shown plays a role in determining viscerotropism. The virusesand methods of the invention are described further, as follows.

One example of a flavivirus that can be used in the invention is yellowfever virus. Mutations can be made in the hinge region of the envelopeof a wild-type infectious clone, e.g., the Asibi infectious clone or aninfectious clone of another wild-type, virulent yellow fever virus, andthe mutants can then be tested in an animal model system (e.g., inhamster and/or monkey model systems) to identify sites affectingviscerotropism. Reduction in viscerotropism is judged by, for example,detection of decreased viremia and/or liver injury in the model system(see below for additional details). One or more mutations found todecrease viscerotropism of the wild-type virus are then introduced intoa vaccine strain (e.g., YF17D), and these mutants are tested in ananimal model system (e.g., in a hamster and/or a monkey model system) todetermine whether the resulting mutants have decreased viscerotropism.Mutants that are found to have decreased viscerotropism can then be usedas new vaccine strains that have increased safety, due to decreasedlevels of viscerotropism.

Additional flaviviruses that can be used in the invention include othermosquito-borne flaviviruses, such as Japanese encephalitis, Dengue(serotypes 1-4), Murray Valley encephalitis, St. Louis encephalitis,West Nile, Kunjin, Rocio encephalitis, and Ilheus viruses; tick-borneflaviviruses, such as Central European encephalitis, Siberianencephalitis, Russian Spring-Summer encephalitis, Kyasanur ForestDisease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi,Absettarov, Hansalova, Apoi, and Hypr viruses; as well as viruses fromthe Hepacivirus genus (e.g., Hepatitis C virus). All of these viruseshave some propensity to infect visceral organs. The viscerotropism ofthese viruses may not cause dysfunction of vital visceral organs, butthe replication of virus in these organs can cause viremia and thuscontribute to invasion of the central nervous system. Decreasing theviscerotropism of these viruses by mutagenesis can thus reduce theirabilities to invade the brain and to cause encephalitis.

In addition to the viruses listed above, as well as other flaviviruses,chimeric flaviviruses that include one or more mutations that decreaseviscerotropism are included in the invention. These chimeras can consistof a flavivirus (i.e., a backbone flavivirus) in which a structuralprotein (or proteins) has been replaced with a corresponding structuralprotein (or proteins) of a second virus (i.e., a test or a predeterminedvirus, such as a flavivirus). For example, the chimeras can consist of abackbone flavivirus (e.g., a yellow fever virus) in which the prM and Eproteins of the flavivirus have been replaced with the prM and Eproteins of the second, test virus (e.g., a dengue virus (1-4), Japaneseencephalitis virus, West Nile virus, or another virus, such as any ofthose mentioned herein)(the E protein of which has a hinge regionmutation as described herein). The chimeric viruses can be made from anycombination of viruses. Preferably, the virus against which immunity issought is the source of the inserted structural protein(s).

A specific example of a chimeric virus that can be included in thevaccines of the invention is the yellow fever human vaccine strain,YF17D, in which the prM protein and the E protein have been replacedwith the prM protein and the E protein (including a hinge mutation asdescribed herein) of another flavivirus, such as a Dengue virus(serotype 1, 2, 3, or 4), Japanese encephalitis virus, West Nile virus,St. Louis encephalitis virus, Murray Valley encephalitis virus, or anyother flavivirus, such as one of those listed above. For example, thefollowing chimeric flaviviruses, which were deposited with the AmericanType Culture Collection (ATCC) in Manassas, Va., U.S.A. under the termsof the Budapest Treaty and granted a deposit date of Jan. 6, 1998, canbe used to make viruses of the invention: Chimeric Yellow Fever17D/Dengue Type 2 Virus (YF/DEN-2; ATCC accession number ATCC VR-2593)and Chimeric Yellow Fever 17D/Japanese Encephalitis SA14-14-2 Virus(YF/JE A1.3; ATCC accession number ATCC VR-2594). Details of makingchimeric viruses that can be used in the invention are provided, forexample, in International applications PCT/US98/03894 andPCT/US00/32821; and Chambers et al., J. Virol. 73:3095-3101, 1999, eachof which is incorporated by reference herein in its entirety.

As is noted above, mutations that are included in the viruses of thepresent invention decrease viscerotropism. In one example, thesemutations are present in the hinge region of the flavivirus envelopeprotein. The polypeptide chain of the envelope protein folds into threedistinct domains: a central domain (domain I), a dimerization domain(domain II), and an immunoglobulin-like module domain (domain III). Thehinge region is present between domains I and II and, upon exposure toacidic pH, undergoes a conformational change (hence the designation“hinge”) involved in the fusion of viral and endosomal membranes, aftervirus uptake by receptor-mediated endocytosis. Numerous envelope aminoacids are present in the hinge region including, for example, aminoacids 48-61, 127-131, and 196-283 of yellow fever virus (Rey et al.,Nature 375:291-298, 1995). Any of these amino acids, or closelysurrounding amino acids (and corresponding amino acids in otherflavivirus envelope proteins), can be mutated according to theinvention, and tested for a resulting decrease in viscerotropism.Mutations can be made in the hinge region using standard methods, suchas site-directed mutagenesis. One example of the type of mutationpresent in the viruses of the invention is substitutions, but othertypes of mutations, such as deletions and insertions, can be used aswell. In addition, as is noted above, the mutations can be presentsingly or in the context of one or more additional mutations.

The viruses (including chimeras) of the present invention can be madeusing standard methods in the art. For example, an RNA moleculecorresponding to the genome of a virus can be introduced into primarycells, chick embryos, or diploid cell lines, from which (or thesupernatants of which) progeny virus can then be purified. Anothermethod that can be used to produce the viruses employs heteroploidcells, such as Vero cells (Yasumura et al., Nihon Rinsho 21, 1201-1215,1963). In this method, a nucleic acid molecule (e.g., an RNA molecule)corresponding to the genome of a virus is introduced into theheteroploid cells, virus is harvested from the medium in which the cellshave been cultured, harvested virus is treated with a nuclease (e.g., anendonuclease that degrades both DNA and RNA, such as Benzonase™; U.S.Pat. No. 5,173,418), the nuclease-treated virus is concentrated (e.g.,by use of ultrafiltration using a filter having a molecular weightcut-off of, e.g., 500 kDa), and the concentrated virus is formulated forthe purposes of vaccination. Details of this method are provided in U.S.Patent Application Ser. No. 60/348,565, filed Jan. 15, 2002, which isincorporated herein by reference.

The viruses of the invention can be administered as primary prophylacticagents in adults or children at risk of infection, or can be used assecondary agents for treating infected patients. For example, in thecase of yellow fever/dengue chimeras, the vaccines can be used in adultsor children at risk of Dengue infection, or can be used as secondaryagents for treating Dengue-infected patients. Examples of patients whocan be treated using the dengue-related vaccines and methods of theinvention include (i) children in areas in which Dengue is endemic, suchas Asia, Latin America, and the Caribbean, (ii) foreign travelers, (iii)military personnel, and (iv) patients in areas of a Dengue epidemic.Moreover, inhabitants of regions into which the disease has beenobserved to be expanding (e.g., Argentina, Chile, Australia, parts ofAfrica, southern Europe, the Middle East, and the southern UnitedStates), or regions in which it may be observed to expand in the future(e.g., regions infested with Aedes aegypti), can be treated according tothe invention.

Formulation of the viruses of the invention can be carried out usingmethods that are standard in the art. Numerous pharmaceuticallyacceptable solutions for use in vaccine preparation are well known andcan readily be adapted for use in the present invention by those ofskill in this art. (See, e.g., Remington's Pharmaceutical Sciences(18^(th) edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton,Pa.) In two specific examples, the viruses are formulated in MinimumEssential Medium Earle's Salt (MEME) containing 7.5% lactose and 2.5%human serum albumin or MEME containing 10% sorbitol. However, theviruses can simply be diluted in a physiologically acceptable solution,such as sterile saline or sterile buffered saline. In another example,the viruses can be administered and formulated, for example, in the samemanner as the yellow fever 17D vaccine, e.g., as a clarified suspensionof infected chicken embryo tissue, or a fluid harvested from cellcultures infected with the chimeric yellow fever virus.

The vaccines of the invention can be administered using methods that arewell known in the art, and appropriate amounts of the vaccinesadministered can be readily be determined by those of skill in the art.For example, the viruses of the invention can be formulated as sterileaqueous solutions containing between 10² and 10⁷ infectious units (e.g.,plaque-forming units or tissue culture infectious doses) in a dosevolume of 0.1 to 1.0 ml, to be administered by, for example,intramuscular, subcutaneous, or intradermal routes. In addition, becauseflaviviruses may be capable of infecting the human host via the mucosalroutes, such as the oral route (Gresikova et al., “Tick-borneEncephalitis,” In The Arboviruses, Ecology and Epidemiology, Monath(ed.), CRC Press, Boca Raton, Fla., 1988, Volume IV, 177-203), theviruses can be administered by mucosal routes as well. Further, thevaccines of the invention can be administered in a single dose or,optionally, administration can involve the use of a priming dosefollowed by a booster dose that is administered, e.g., 2-6 months later,as determined to be appropriate by those of skill in the art.

Optionally, adjuvants that are known to those skilled in the art can beused in the administration of the viruses of the invention. Adjuvantsthat can be used to enhance the immunogenicity of the viruses include,for example, liposomal formulations, synthetic adjuvants, such as (e.g.,QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine.Although these adjuvants are typically used to enhance immune responsesto inactivated vaccines, they can also be used with live vaccines. Inthe case of a virus delivered via a mucosal route, for example, orally,mucosal adjuvants such as the heat-labile toxin of E. coli (LT) ormutant derivations of LT can be used as adjuvants. In addition, genesencoding cytokines that have adjuvant activities can be inserted intothe viruses. Thus, genes encoding cytokines, such as GM-CSF, IL-2,IL-12, IL-13, or IL-5, can be inserted together with foreign antigengenes to produce a vaccine that results in enhanced immune responses, orto modulate immunity directed more specifically towards cellular,humoral, or mucosal responses.

In the case of Dengue virus, against which optimal vaccination caninvolve the induction of immunity against all four of the dengueserotypes, the chimeric viruses of the present invention can be used inthe formulation of tetravalent vaccines. Any or all of the chimeras usedin such tetravalent formulations can include a mutation that decreasesviscerotropism, as is described herein. The chimeras can be mixed toform tetravalent preparations at any point during formulation, or can beadministered in series. In the case of a tetravalent vaccine, to reducethe possibility of viral interference and thus to achieve a balancedimmune response, the amounts of each of the different chimeras presentin the administered vaccines can vary. Briefly, in one example of such aformulation, at least 5 fold less of the Dengue-2 chimera (e.g., 10, 50,100, 200, or 500 fold less) is used relative to the other chimeras. Inthis example, the amounts of the Dengue-1, Dengue-3, and Dengue-4chimeras can be equivalent or can vary. In another example, the amountsof Dengue-4 and/or Dengue 1 virus can be decreased as well. For example,in addition to using less Dengue-2 chimera, at least 5 fold less of theDengue-4 chimera (e.g., 10, 50, 100, 200, or 500 fold less) can be usedrelative to the Dengue-1 and Dengue-3 chimeras; at least 5 fold less ofthe Dengue-1 chimera (e.g., 10, 50, 100, 200, or 500 fold less) can beused relative to the Dengue-3 and Dengue-4 chimeras; or at least 5 foldless of the Dengue-1 and Dengue-4 chimeras can be used relative to theDengue-3 chimera. It may be particularly desirable, for example, todecrease the amount of Dengue-1 chimera relative to the amounts ofDengue-3 and/or Dengue-4 chimeras when the E204/E202 mutation describedherein is not included in the chimera.

Details of the characterization of one example of a mutation included inthe invention, which occurs at position 279 of the envelope protein of ayellow fever/Japanese encephalitis chimera, are provided below. Alsoprovided below are details concerning yellow fever/dengue viruschimeras, in which dengue virus envelope proteins include one or moremutations that decrease viscerotropism. In one example of such amutation, the lysine at position 204 of the envelope protein ofDengue-1, Dengue-2, or Dengue-4, or the lysine at position 202 of theenvelope protein of Dengue-3, which is two amino acids shorter than theenvelope proteins of the other Dengue serotypes, is substituted ordeleted. This lysine can be, for example, substituted with arginine.Other residues near envelope amino acid 204 (202 for Dengue-3) can alsobe mutated to achieve decreased viscerotropism. For example, any ofamino acids 200-208 or combinations of these amino acids can be mutated.Specific examples include the following: position 202 (K) of Dengue-1;position 202 (E) of Dengue-2; position 200 of Dengue-3 (K); andpositions 200 (K), 202 (K), and 203(K) of Dengue-4. These residues canbe substituted with, for example, arginine.

Experimental Results I. Yellow Fever/Japanese Encephalitis ChimeraIncluding a Hinge Region Mutation

Summary

A chimeric yellow fever (YF)-Japanese encephalitis (JE) vaccine(ChimeriVax™-JE) was constructed by insertion of the prM-E genes fromthe attenuated JE SA14-14-2 vaccine strain into a full-length cDNA cloneof YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to theemergence of a small-plaque virus containing a single Met→Lys amino acidmutation at E279, reverting this residue from the SA14-14-2 to thewild-type amino acid. A similar virus was also constructed bysite-directed mutagenesis. The E279 mutation is located in a beta-sheetin the hinge region of the E protein, which is responsible for apH-dependent conformational change during virus penetration from theendosome into the cytoplasm of an infected cell. In independenttransfection-passage studies in FRhL or Vero cells, mutations appearedmost frequently in hinge 4 (bounded by amino acids E266 to E284),reflecting genomic instability in this functionally important region.The E279 reversion caused a significant increase in neurovirulence, asdetermined by LD50 and survival distribution in suckling mice and byhistopathology in rhesus monkeys. Based on sensitivity and comparabilityof results with monkeys, the suckling mouse is an appropriate host forsafety testing of flavivirus vaccine candidates for neurotropism. TheE279 Lys virus was restricted with respect to extraneural replication inmonkeys, as viremia and antibody levels (markers of viscerotropism) weresignificantly reduced as compared to E279 Met virus.

Background

The study of chimeric viruses has afforded new insights into themolecular basis of virulence and new prospects for vaccine development.For example, molecular clones of positive-strand alphaviruses(Morris-Downes et al., Vaccine 19:3877-3884, 2001; Xiong et al., Science243:1188-1191, 1991) and flaviviruses (Bray et al., Proc. Natl. Acad.Sci. U.S.A. 88:10342-10346, 1991; Chambers et al., J. Virol.73:3095-3101, 1999; Guirakhoo et al., J. Virol. 75:7290-7304, 2001;Huang et al., J. Virol. 74:3020-3028, 2000) have been modified byinsertion of structural genes encoding the viral envelope anddeterminants involved in neutralization, cell attachment, fusion, andinternalization. The replication of these chimeric viruses is controlledby nonstructural proteins and the non-coding termini expressed by theparental strain, while the structural proteins from the donor genesafford specific immunity. The biological characteristics of chimericviruses are determined by both the donor and recipient virus genes. Bycomparing constructs with nucleotide sequence differences across thedonor genes, it is possible to dissect out the functional roles ofindividual amino acid residues in virulence and attenuation.

Using a chimeric yellow fever (YF) virus that incorporated the prM-Egenes from an attenuated strain (SA14-14-2) of Japanese encephalitis(JE), a detailed examination was made of the role of 10 amino acidmutations that distinguished the attenuated JE virus from virulent,wild-type JE Nakayama virus (Arroyo et al., J. Virol. 75:934-942, 2001).The virulence factors were defined by reverting each mutation singly oras clusters to the wild-type sequence and determining the effects onneurovirulence for young adult mice inoculated by the intracerebral (IC)route with 10⁴ plaque-forming units (PFU). All of the single-siterevertant viruses remained highly attenuated, and reversions at 3 or 4residues were required to restore a neurovirulent phenotype. Only onesingle-site revertant (E279 Met→Lys) showed any evidence of a change invirulence, with 1 of 8 animals succumbing after IC inoculation.

In order to explore further the functional role of the E279 determinant,we compared chimeric YF/JE viruses that differed at this amino acidresidue for their abilities to cause encephalitis in suckling mice andmonkeys. IC inoculation of monkeys is routinely used as a test forsafety of flavivirus and other live vaccines, and quantitativepathological examination of brain and spinal cord tissue provides asensitive method for distinguishing strains of the same virus withsubtle differences in neurovirulence (Levenbook et al., J. Biol. Stand.15: 305-313, 1987). Suckling mice provide a more sensitive model thanolder animals, since susceptibility to neurotropic flaviviruses isage-dependent (Monath et al., J. Virol. 74:1742-1751, 2000). The resultsconfirmed that the single Met→Lys amino acid mutation at E279 conferredan increase in neurovirulence. This mutation is located in the ‘hinge’region of the E protein, which is responsible for a pH-dependentconformational change during virus penetration from the endosome intothe cytoplasm of an infected cell (Reed et al., Am. J. Hyg. 27:493-497,1938). Importantly, the suckling mouse was shown to predict thevirulence profile in rhesus monkeys. Based on the detection of a changein neurovirulence conferred by a point mutation, we propose that thesuckling mouse is an appropriate host for safety testing of flavivirusvaccine candidates for neurotropism.

While enhancing neurovirulence, the E279 mutation appeared to have theopposite effect on viscerotropism, as measured by decreased viremia andantibody response in monkeys, accepted markers of this viral trait (Wanget al., J. Gen. Virol. 76:2749-2755, 1995).

Materials and Methods

Viruses

Development of the ChimeriVax™-JE vaccine began by cloning a cDNA copyof the entire 11-kilobase genome of YF 17D virus (Chambers et al., J.Virol. 73:3095-3101, 1999). To accomplish this, YF 17D genomic sequenceswere propagated in two plasmids, which encode the YF sequences fromnucleotide (nt) 1-2276 and 8279-10,861 (plasmid YF5′3′IV), and from1373-8704 (plasmid YFM5.2), respectively. Full-length cDNA templateswere generated by ligation of appropriate restriction fragments derivedfrom these plasmids. YF sequences within the YF 5′31V and YFM5.2plasmids were replaced by the corresponding JE (SA14-14-2) pr-MEsequences, resulting in the generation of YF5′3′IV/JE (prM-E′) andYFM5.2/JE (E′-E) plasmids. These plasmids were digested sequentiallywith restriction endonucleases NheI and BspEI. Appropriate fragmentswere ligated with T4 DNA ligase, cDNA was digested with XhoI enzyme toallow transcription, and RNA was produced from an Sp6 promoter.Transfection of diploid fetal rhesus lung (FRhL) cells with full-lengthRNA was performed by electroporation. Supernatant containing virus washarvested when cytopathic effect was observed (generally day 3),clarified by low-speed centrifugation and sterile-filtered at 0.22 μm.Fetal bovine serum (FBS) 50% v/v final concentration was added as astabilizer. The virus was titrated by plaque assay in Vero cells, aspreviously described (Monath et al., Vaccine 17:1869-1882, 1999). Thechimeric virus was sequentially passed in FRhL or Vero cells (Vero-PM,Aventis Pasteur, Marcy l'Étoile, France) at a multiplicity of infectionof approximately 0.001. Commercial yellow fever 17D vaccine (YF-VAX®)was obtained from Aventis-Pasteur (formerly Pasteur-Mérieux-Connaught),Swiftwater, Pa.

Site-directed Mutagenesis

Virus containing a single-site Met→Lys reversion at residue E279 wasgenerated by oligo-directed mutagenesis as described (Arroyo et al., J.Virol. 75:934-942, 2001). Briefly, a plasmid (pBS/JE SA14-14-2)containing the JE SA-14-14-2 E gene region from nucleotides 1108 to 2472(Cecilia et al., Virology 181:70-77, 1991) was used as template forsite-directed mutagenesis. Mutagenesis was performed using theTransformer site-directed mutagenesis kit (Clontech, Palo Alto, Calif.)and oligonucleotide primers synthesized at Life Technologies (GrandIsland, N.Y.). Plasmids were sequenced across the E region to verifythat the only change was the engineered mutation. A region encompassingthe E279 mutation was then subcloned from the pBS/JE plasmid intopYFM5.2/JE SA14-14-2 (Cecilia et al., Virology 181:70-77, 1991) usingthe NheI and EheI (KasI) restriction sites. Assembly of full-length DNAand SP6 transcription were performed as described above; however, RNAtransfection of Vero cells was performed using Lipofectin (Gibco/BRL).

Sequencing

RNA was isolated from infected monolayers by Trizol® (LifeTechnologies). Reverse transcription was performed with Superscript IIReverse Transcriptase (RT) and a long-RT protocol (Life Technologies),followed by RNaseH treatment (Promega) and long-PCR (XL PCR,Perkin-Elmer/ABI). RT, PCR, and sequencing primers were designed usingYF17D strain sequence (GeneBank Accession number K02749) andJE-SA14-14-2 strain sequence (GeneBank Accession number D90195) asreferences. PCR products were gel-purified (Qiaquick gel-extraction kitfrom Qiagen) and sequenced using Dye-Terminator dRhodamine sequencingreaction mix (Perkin-Elmer/ABI). Sequencing reactions were analyzed on amodel 310 Genetic Analyzer (Perkin-Elmer/ABI) and DNA sequences wereevaluated using Sequencher 3.0 (GeneCodes) software.

Plaque Assays and Neutralization Tests

Plaque assays were performed in 6 well plates of monolayer cultures ofVero cells. After adsorption of virus for 1 hour incubation at 37° C.,the cells were overlaid with agarose in nutrient medium. On day 4, asecond overlay was added containing 3% neutral red. Serum-dilution,plaque-reduction neutralization tests were performed as previouslydescribed (Monath et al., Vaccine 17:1869-1882, 1999).

Weaned Mouse Model

Groups of 8 to 10 female 4 week old ICR mice (Taconic Farms, Inc.Germantown, N.Y.) were inoculated IC with 30 μL of chimeric YF/JESA14-14-2 (ChimeriVax™-JE) constructs with (dose 4.0 log₁₀ PFU in) orwithout (3.1 log₁₀ PFU) the E279 mutation. An equal number of mice wereinoculated with YF-VAX® or diluent. Mice were followed for illness anddeath for 21 days.

Suckling Mouse Model

Pregnant female ICR mice (Taconic Farms) were observed throughparturition in order to obtain litters of suckling mice of exact age.Suckling mice from multiple litters born within a 48 hour interval werepooled and randomly redistributed to mothers in groups of up to 121mice. Litters were inoculated IC with 20 μL of serial tenfold dilutionsof virus and followed for signs of illness and death for 21 days. Thevirus inocula were back-titrated. 50% lethal dose (LD₅₀) values werecalculated by the method of Reed and Muench (Morris-Downes et al.,Vaccine 19:3877-3884, 2001). Univariate survival distributions wereplotted and compared by log rank test.

Monkey Model

The monkey neurovirulence test was performed as described by Levenbooket al. (Levenbook et al., J. Biol. Stand. 15: 305-313, 1987) andproscribed by WHO regulations for safety testing YF 17D seed viruses(Wang et al., J. Gen. Virol. 76:2749-2755, 1995). This test haspreviously been applied to the evaluation of ChimeriVax™-JE vaccines,and results of tests on FRhL₃ virus were described (Monath et al., Curr.Drugs—Infect. Dis., 1:37-50; 2001; Monath et al., Vaccine 17:1869-1882,1999). Tests were performed at Sierra Biomedical Inc. (Sparks, Nev.),according to the U.S. Food and Drug Administration Good LaboratoryPractice (GLP) regulations (21 C.F.R., Part 58). On Day 1, ten (5 male,5 female) rhesus monkeys weighing 3.0-6.5 kg received a singleinoculation of 0.25 mL undiluted ChimeriVax™-JE virus with or withoutthe E279 Met→Lys mutation or YF-VAX® into the frontal lobe of the brain.Monkeys were evaluated daily for clinical signs and scored as 0 (nosigns), 1 (rough coat, not eating), 2 (high-pitched voice, inactive,slow moving, 3 (shaky movements, tremors, incoordination, limbweakness), and 4 (inability to stand, limb paralysis, death). Theclinical score for each monkey is the mean of the animal's daily scores,and the clinical score for the treatment group is the arithmetic mean ofthe individual clinical scores. Viremia levels were measured by plaqueassay in Vero cells using sera collected on days 2-10. On day 31,animals were euthanized, perfused with isotonic saline-5% acetic acidfollowed by neutral-buffered 10% formalin, and necropsies wereperformed. Brains and spinal cords were fixed, sectioned and stainedwith gallocyanin. Neurovirulence was assessed by the presence andseverity of lesions in various anatomical formations of the centralnervous system. Severity was scored within each tissue block using thescale specified by WHO (Wang et al., J. Gen. Virol. 76:2749-2755, 1995):

Grade 1: Minimal: 1-3 small focal inflammatory infiltrates. A fewneurons may be changed or lost.

Grade 2: Moderate: more extensive focal inflammatory infiltrates.Neuronal changes or loss affects not more than one-third of neurons.

Grade 3: Severe: neuronal changes or loss affecting 33-90% of neurons;moderate focal or diffuse inflammatory changes

Grade 4: Overwhelming; more than 90% of neurons are changed or lost,with variable but frequently severe inflammatory infiltration

Structures involved in the pathologic process most often and withgreatest severity were designated ‘target areas,’ while those structuresdiscriminating between wild-type JE virus and ChimeriVax™-JE weredesignated ‘discriminator areas.’ The substantia nigra constituted the‘target area’ and the caudate nucleus, globus pallidus, putamen,anterior/medial thalamic nucleus, lateral thalamic nucleus, and spinalcord (cervical and lumbar enlargements) constituted ‘discriminatorareas’ (Monath et al., Curr. Drugs Infect. Dis., 1:37-50, 2001), aspreviously shown for YF 17D (Levenbook et al., J. Biol. Stand.15:305-313, 1987). All neuropathological evaluations were done by asingle, experienced investigator who was blinded to the treatment code.Separate scores for target area, discriminator areas, andtarget+discriminator areas were determined for each monkey, and testgroups compared with respect to average scores. Other areas of thebrainstem (nuclei of the midbrain in addition to substantia nigra; pons;medulla; and cerebellum) and the leptomeninges were also examined.Statistical comparisons of mean neuropathological scores (for the targetarea, discriminator areas, and target+discriminator areas) wereperformed by Student's t test, 2-tailed. In addition toneuropathological examination, the liver, spleen, adrenal glands, heart,and kidneys were examined for pathologic changes by light microscopy.

Genome Stability

To ascertain the genetic stability of the YF/JE chimeric virus, and tosearch for ‘hot spots’ in the vaccine genome that are susceptible tomutation, multiple experiments were performed in which RNA was used totransfect cells and the progeny virus serially passaged in vitro, withpartial or complete genomic sequencing performed at low and high passagelevels. Passage series were performed starting with the transfectionstep in FRhL or Vero-PM cells. Serial passage of the virus was performedat low MOI in cell cultures grown in T25 or T75 flasks. At selectedpassage levels, duplicate samples of viral genomic RNA were extracted,reverse-transcribed, amplified by PCR, and the prM-E region or fullgenomic sequence determined.

Results

Generation of Single-site Mutant Viruses by Empirical Passage

The chimeric YF/JESA14-14-2 (ChimeriVax™-JE) virus recovered fromtransfected FRhL cells (FRhL₁) was passed sequentially in fluid culturesof these cells at an MOI of approximately 0.001. As is described below,at passage 4 we noted a change in plaque morphology, which wassubsequently shown to be associated with a T→G transversion atnucleotide 1818 resulting in an amino acid change (Met→Lys) at position279 of the E protein.

Plaques were characterized at each passage level and classified into 3categories based on their sizes measured on day 6 (large, L˜>1.0 mm,medium, M˜0.5-1 mm, and small, S˜<0.5 mm). The plaque size distributionwas determined by counting 100 plaques. FRhL₃ (3^(rd) ) passage) viruscontained 80-94% L and 6-20% S plaques. At FRhL₅ (5^(th) passage), achange in plaque size was detected, with the emergence of S plaquescomprising >85% of the total plaque population (FIG. 1). The FRhL₄ viruswas intermediate, with 40% large and 60% small plaques. Full genomicsequencing of the FRhL₅ virus demonstrated a single mutation at E279.The full genome consensus sequence of the FRhL₅ chimera, with carefulinspection for codon heterogeneity, confirmed that this was the onlydetectable mutation present in the virus. The full genome consensussequence of the FRhL₃ virus revealed no detectable mutations compared tothe parental YF/JESA14-14-2 chimeric virus (Arroyo et al., J. Virol.75:934-942, 2001) (Table 1).

Ten large, medium, and small plaques were picked from FRhL₃, ₋₄ and ₋₅,and amplified by passage in fluid cultures of FRhL cells. Afteramplification, the supernatant fluid was plagued on Vero cells. Attemptsto isolate the S plaque phenotype from FRhL₃ failed and all isolated Lor S size plaques produced a majority of L plaques after one round ofamplification in FRhL cells. At the next passage (FRhL₄), where 60% ofplaques were of small size, it was possible to isolate these plaques byamplification in FRhL cells. At FRhL₅, the majority of plaques (85-99%)were of small size, and amplification of both L and S individual plaquesresulted in majority of S size. Sequencing the prM-E genes of the S andL plaque phenotypes from FRhL₃ revealed identical sequences to theparent SA14-14-2 genes used for construction of ChimeriVax™-JE, whereasS plaques isolated from either FRhL₄ or FRhL₅ virus revealed themutation (Met→Lys) at E279.

Animal Protocols

All studies involving mice and nonhuman primates were conducted inaccordance with the USDA Animal Welfare Act (9 C.F.R., Parts 1-3) asdescribed in the Guide for Care and Use of Laboratory Animals.

Virulence for Weaned Mice

Ten female ICR mice 4 weeks of age were inoculated IC with approximately3.0 log₁₀ PFU of FRhL₃, ₋₄, or ₋₅ virus in separate experiments; in eachstudy 10 mice received an equivalent dose (approximately 3.3 log₁₀ PFU)of commercial yellow fever vaccine (YF-VAX®, Aventis Pasteur, SwiftwaterPa.). None of the mice inoculated with chimeric viruses showed signs ofillness or died, whereas 70-100% of control mice inoculated with YF-VAX®developed paralysis or died. In another experiment, 8 mice wereinoculated IC with FRhL₅ (3.1 log₁₀ PFU) or the YF/JE single-site E279revertant (4.0 log₁₀ PFU) and 9 mice received YF-VAX® (2.3 log₁₀ PFU).None of the mice inoculated with the chimeric constructs became ill,whereas 6/9 (67%) of mice inoculated with YF-VAX® died.

Virulence for Suckling Mice

Two separate experiments were performed in which YF/JESA14-14-2 chimericviruses with and without the E279 mutation were inoculated IC at gradeddoses into suckling mice (Table 2). YF-VAX® was used as the referencecontrol in these experiments. LD₅₀ and average survival times (AST) weredetermined for each virus.

In the first experiment using mice 8.6 days old, FRhL₅ virus containingthe single site reversion (Met→Lys) at E279 was neurovirulent, with alog₁₀ LD₅₀ of 1.64 whereas the FRhL₃ virus lacking this mutation wasnearly a virulent, with only 1 of 10 mice dying in the highest dosegroups (Table 2). At the highest dose (approximately 3 log₁₀ PFU), theAST of the FRhL₅ virus was shorter (10.3 days) than that of the FRhL₃virus (15 days).

A second experiment was subsequently performed to verify statisticallythat a single site mutation in the E gene is detectable byneurovirulence test in suckling mice. In this experiment outbred mice 4days of age were inoculated IC with graded doses of ChimeriVax™-JE FRhL₃(no mutation), ChimeriVax™-JE FRhL₅ (E279 Met→Lys), or a YF/JE chimerain which a single mutation E279 (Met→Lys) was introduced at bysite-directed mutagenesis (Arroyo et al., J. Virol. 75:934-942, 2001).The LD₅₀ values of the two viruses containing the E279 mutationwere >10-fold lower than the FRhL₃ construct without the mutation (Table2) indicating that the E279 Met→Lys mutation increased theneurovirulence of the chimeric virus. There were statisticallysignificant differences between the viruses in the survivaldistributions (FIG. 2). At the lowest dose (˜0.7 log₁₀ PFU), the YF/JEchimeric viruses were significantly less virulent than YF-VAX® (log rankp<0.0001). The viruses with the E279 Met→Lys mutation had similarsurvival curves that differed from the FRhL3 virus no mutation), but thedifference did not reach statistical significance (log rank p=0.1216).However, at higher doses (˜1.7 and ˜2.7 log₁₀ PFU), the survivaldistributions of the E279 mutant viruses were significantly differentfrom FRhL₃ virus.

Analysis of mortality ratio by virus dose revealed similar slopes andparallel regression lines (FIG. 3). The FRhL₅ virus was 18.52 times morepotent (virulent) than FRhL₃ (95% fiducial limits 3.65 and 124.44,p<0.0001).

Monkey Neurovirulence Test

None of the 20 monkeys inoculated with ChimeriVax™-JE FRhL₃ or FRhL₅viruses developed signs of encephalitis, whereas 4/10 monkeys inoculatedwith YF-VAX® developed grade 3 signs (tremors) between days 15-29, whichresolved within 6 days of onset. Mean and maximum mean clinical scoreswere significantly higher in the YF-VAX® group than in the twoChimeriVax™-JE groups. There was no difference in clinical score betweengroups receiving ChimeriVax™-JE viruses with and without the E279mutation (Table 3).

There were no differences in weight changes during the experimentbetween treatment groups. Pathological examination revealed noalterations of liver, spleen, kidney, heart, or adrenal glandsattributable to the viruses, and no differences between treatmentgroups.

Histopathologic examination of the brain and spinal cord revealedsignificantly higher lesion scores for monkeys inoculated with YF-VAX®than for ChimeriVax™-JE virus FRhL₃ and FRhL₅ (Table 3). The combinedtarget+discriminator scores (±SD) for YF-VAX® was 1.17 (±0.47). Thescores for the ChimeriVax™-JE FRhL₃ (E279 Met) and FRhL₅ (E279 Lys) were0.29 (±0.20), (p=0.00014 vs. YF-VAX®) and 0.54 (±0.28), (p=0.00248 vs.YF-VAX®), respectively.

The discriminator area score and combined target+discriminator areascore for ChimeriVax™-JE FRhL₅ containing the Met→Lys reversion at E279were significantly higher than the corresponding scores forChimeriVax™-JE FRhL₃ (Table 3).

The main symptom in monkeys inoculated with YF-VAX® was tremor, whichmay reflect lesions of the cerebellum, thalamic nuclei, or globuspallidus. No clear histological lesions were found in the cerebellarcortex, N. dentatus, or other cerebellar nuclei, whereas inflammatorylesions were present in the thalamic nuclei and globus pallidus in allpositive monkeys.

Interestingly, there was an inverse relationship between neurovirulenceand viscerotropism of the E279 revertant, as reflected by viremia. TheWHO monkey neurovirulence test includes quantitation of viremia as ameasure of viscerotropism (World Health Organization, “Requirements foryellow fever vaccine,” Requirements for Biological Substances No. 3,revised 1995, WHO Tech. Rep. Ser. 872, Annex 2, Geneva: WHO, 31-68,1998). This is rational, based on observations that intracerebralinoculation results in immediate seeding of extraneural tissues(Theiler, “The Virus,” In Strode (ed.), Yellow Fever, McGraw Hill, NewYork, N.Y., 46-136, 1951). Nine (90%) of 10 monkeys inoculated withYF-VAX® and 8 (80%) of 10 monkeys inoculated with ChimeriVax™-JE FRhL₃became viremic after IC inoculation. The level of viremia tended to behigher in the YF-VAX® group than in the ChimeriVax™-JE FRhL₃ group,reaching significance on Day 4. In contrast, only 2 (20%) of the animalsgiven FRhL₅ virus (E279 Met→Lys) had detectable, low-level viremias(Table 4), and the mean viremia was significantly lower than FRhL₃ viruson days 3 and 4 (and nearly significant on day 5). Thus, the FRhL₅revertant virus displayed increased neurovirulence, but decreasedviscerotropism compared to FRhL₃ virus. Sera from monkeys inoculatedwith ChimeriVax™-JE FRhL₃ and FRhL₅ were examined for the presence ofplaque size variants. Only L plaques were observed in sera from monkeysinoculated with the FRhL₃, whereas the virus in blood of monkeysinoculated with FRhL₅ had the appropriate S plaque morphology.

Immunogenicity

All monkeys in all three groups developed homologous neutralizingantibodies 31 days post-inoculation to yellow fever (YF-VAX® group) orChimeriVax™-JE (ChimeriVax™ groups), with the exception of 1 animal(FRhL₅, RAK22F), which was not tested due to sample loss. However, thegeometric mean antibody titer (GMT) was significantly higher in themonkeys inoculated with FRhL₃ (GMT 501) than with FRhL₅ (GMT 169,p=0.0386, t-test).

Genome Stability

Two separate transfections of ChimeriVax™-JE RNA were performed in eachof two cell strains, FRhL and Vero, and progeny viruses were passed asis shown in FIG. 4. The FRhL passage series B resulted in appearance ofthe E279 reversion at FRhL₄ as described above. Interestingly, aseparate passage series (A) in FRhL cells also resulted in theappearance of a mutation (Thr→Lys) in an adjacent residue at E281, andone of the passage series in Vero cells resulted in a Val→Lys mutationat E271. Other mutations selected in Vero cells were in domain III orwithin the transmembrane domain. All viruses containing mutations shownin FIG. 2 were evaluated in the adult mouse neurovirulence test and werefound to be avirulent.

II. Yellow Fever/Dengue Chimera Including Hinge Region Mutation

Summary

ChimeriVax™-DEN1 virus was produced using the prME genes of a wild typestrain of dengue 1 virus [(Puo359) isolated in 1980 in Thailand]inserted into the yellow fever virus (strain 17D) backbone (Guirakhoo etal., J. Virol. 75:7290-7304, 2001). During production of a Pre-MasterSeed virus for ChimeriVax™-DEN1 in Vero cells, a clone (clone E)containing a single nucleotide change from A to G at position 1590,which resulted in an amino acid substitution from K to R at position 204on the envelope protein E, was isolated and plaque purified. The virusexhibited attenuation for 4-day-old suckling mice and produced a lowerviremia (viscerotropism) than its parent (non-mutant) virus wheninoculated by subcutaneous route into monkeys. Another clone (clone J-2)without mutation was selected, plaque purified, and used to produce aPMS virus stock at passage 7 (P7). This virus did not undergo anymutations when passaged under laboratory conditions up to P10 in Verocells. However, upon one passage under cGMP conditions to produce aMaster Seed virus (P8) from PMS stock, the same mutation at position1590 (A to G) emerged. Similar to clone E, the P8 virus produced largerplaques than P7 virus and was attenuated for suckling mice. The E204position, which is conserved in all dengue viruses, can thus bemanipulated in ChimeriVax™-DEN (serotypes 1-4) viruses to achieve abalance between attenuation and immunogenicity of the vaccine candidatesfor humans.

Results and Discussion

Production of Pre-Master Seeds for ChimeriVax-DEN1 Viruses

Production of plaque purified Pre-Master Seed (PMS) viruses for DEN1 wascarried out as follows. Plaque purification was started with the virusat Passage 2 (P2) post RNA transfection. Two PMS viruses (uncloned at P2and cloned at P7) were produced in Aventis Vero LS10 cells at passage142 using a qualified cell bank obtained from Aventis at passage 140.Cloned viruses were obtained after 3 rounds of plaque purification andsequenced across the full genome to assure lack of mutations. Generally,if a clone contained an amino acid substitution, it was not used as aPMS virus candidate. Other clones were prepared and sequenced until aclone without mutation was identified, which was then subjected toplaque purification and sequencing.

Sequencing

For sequencing, viral RNA was extracted from each individual virussample (generally 0.25 ml) using TRI-Reagent LS (Molecular ResearchCenter) or Trizol LS (a similar reagent from Gibco) and dissolved in0.20 ml of RNase-free water. The extracted RNA was then used as atemplate for RT-PCR. The entire genome was amplified in five overlappingamplicons of ˜2-3 kb in length (fragments I through V) with the TitanOne-Tube RT-PCR kit (Roche). The RT-PCR fragments were purified usingQIAquick PCR Purification kit (Qiagen) or agarose gel-purified usingQIAquick Gel Extraction kit (Qiagen). Sequencing reactions were doneusing CEQ Dye Terminator Cycle Sequencing kit (Beckman) and a collectionof YF-specific oligonucleotide primers of both positive and negativeorientation to read both strands of the amplicons. Sequencing reactionproducts were purified using DyeEx Spin kit (Qiagen), and resolved witha CEQ2000 automated sequencer (Beckman Coulter). Generated sequencingdata were aligned and analyzed with Sequencher 3.0 (GeneCodes) software.Nucleotide heterogeneities were registered only when a heterogeneoussignal was observed in all chromatograms representing both plus- andminus-strand sequencing reactions.

As is shown in Table 5, the uncloned P2 virus did not have anymutations, but acquired 5 amino acid mutations (heterogeneity) withinthe envelope protein E by P5. Interestingly, the only mutation that wasstable (further selected) at P15 was the 204 mutation. A repeat passageexperiment starting from uncloned P2 virus (up to P15) revealed the samemutation (K to R) at E204 position being selected in Vero cells.

Different clones of ChimeriVax-DEN1 (A-J) were selected by direct plaqueto plaque purification and sequenced at various stages to identifymutations. The most frequent mutation was the E251 (V>F) substitution,which occurred in clones A, B, D, and G followed by E204 (K>R), whichwas found in clones E and F, as well as in uncloned viruses. Themutation at E311 (E>D) was only found in clones C and D. Interestingly,clone J was free from mutations up to P10. However, when a Master Seed(MS) of this virus was produced from P7 (PMS) under cGMP manufacturing,the same substitution at E204 reemerged (only after 1 passage). Thismutation was stable when P20 virus was sequenced (Table 5). Clonescontaining the E204 mutation produced larger plaques (˜2 mm in diameter)than non-mutant viruses (˜1 mm in diameter) (Table 9). The originalconstruct of this virus at Vero P4 (previously shown to produce a lowlevel of viremia in monkeys) also contained the same E204 mutation(Guirakhoo et al., J. Virol. 75:7290-7304, 2001). The role of thismutation in the biology of the virus could not be understood previouslybecause: a) the original construct contained an additional mutation(nucleotide A to G causing an amino acid change from H to R) at M39besides the E204 mutation; b) the neurovirulence of the originalconstruct was evaluated only in 3-4 week old mice, which are notsensitive enough to reveal attenuation of ChimeriVax-DEN1 virus or anyother ChimeriVax™-DEN viruses (Guirakhoo et al., J. Virol. 75:7290-7304,2001); and c) there was no ChimeriVax™-DEN1 virus (without mutation)available for comparison to determine changes in neurovirulence orviscerotropism phenotype of the virus.

Since chimeric viruses are attenuated for 3-4 week old mice, wedeveloped a more sensitive test (using suckling mice of 4-8 days old) totest subtle differences in neurovirulence of different clones.

Mouse Neurovirulence

The mouse neurovirulence test, using 3-4 week old mice, is performed asa release test to ensure that neurovirulence of chimeras does not exceedthat of the virus vector (YF-VAX®) used to construct ChimeriVax™viruses. Because all chimeras constructed so far (with or withoutmutations) are not virulent for adult mice (3-4 weeks old), theseanimals cannot be used to identify subtle changes in neurovirulence ofchimeras associated with single amino acid substitutions. In contrast,suckling mice of 4-10 days of age are more sensitive to minor changes inthe genome of chimeras, which are involved in virulence. In the courseof development of ChimeriVax™-DEN viruses, several mutations wereobserved across the genome of all 4 chimeras (Guirakhoo et al., J.Virol. 75:7290-7304, 2001). These mutations were corrected in allchimeras, and the reconstructed viruses (except for DEN1 chimeras) weresuccessfully evaluated for safety and immunogenicity in monkeys. Due toinstability of DEN1 plasmids, the reconstruction of this chimera(without mutation) was not accomplished on time, and could therefore notbe tested in monkeys. During plaque purification to produce a PMS forDEN1 chimera, 10 different clones (A-J) were sequenced to identify aclone without mutations (Table 5). All but one clone (J) contained 1 or2 mutations within the envelope protein E. Representative clones of DEN1chimeras were evaluated for their neurovirulence using 4 day-oldsuckling mice (Table 6). Animals were inoculated by the i.c. route withtwo 0.02 ml of undiluted, 1:10, or 1:100 dilutions of each chimeric DEN1virus and observed for 21 days. The actual doses were determined by backtitration of inocula in a plaque assay. As is shown in Table 6, allclones except clone E exhibited similar neurovirulence for 4 day-oldmice with average survival times (AST) significantly lower than that ofYF-VAX® (p<0.001 using JMP software, Version 4.0.2). Clone E (E204K>R)was significantly less virulent than all other DEN1 clones (p<0.0001).Interestingly, one of the 2 mutations identified in the original DEN1chimera was the E204 K>R substitution. This virus induced a low level ofviremia (mean peak titer 0.7 log₁₀ PFU/ml) for 1.3 days when inoculatedinto monkeys (Guirakhoo et al., J. Virol. 75:7290-7304, 2001). Clone J,which did not contain any mutations and was shown to be significantlyless virulent than YF-VAX® in 4 days old mice, P=0.001, was selected forproduction of the cGMP MS virus.

Safety and Immunogenicity (Viremia and Neutralizing Antibody Responses)of Chimeric DEN1 Viruses in Monkeys

The effect of the E204 mutation on viscerotropism (viremia) of the viruswas assessed by inoculation of monkeys with ChimeriVax-DEN1 viruses with(clone E, P6) or without (clone J, P7) the E204 mutation. The originalDEN1 chimera (ChimeriVax-DEN-1, uncloned P4, 1999, Group 1) was selectedas a control, because its viremia and immunogenicity profiles hadalready been evaluated in monkeys as a monovalent or a tetravalent(combined with 3 other chimeras) vaccine (Guirakhoo et al., J. Virol.75:7290-7304, 2001).

Groups of 4 rhesus monkeys were inoculated with 5 log₁₀ PFU/0.5 ml ofDEN1 chimeras (Table 7). Viremia was measured (by plaque assay on Verocells) on sera obtained from Day 2 to Day 11 post infection. All monkeysinoculated with DEN1 PMS virus (Group 3) became viremic, whereas 3/4 and2/4 monkeys inoculated with clone E or uncloned viruses, respectively,became viremic (Table 8). The mean peak virus titer (2.5 log₁₀ PFU/ml)and duration (8.5 days) of viremia in Group 3 monkeys (DEN1 PMS) wassignificantly higher (p=0.024 and 0.0002 for peak virus titer andduration, respectively) than Groups 1 and 2. Despite the lack of viremiain some monkeys, all animals developed neutralizing antibody titersagainst homologous viruses. For neutralization assays, sera from eachgroup of monkeys were heat inactivated and mixed with the homologousvirus (the same virus that had been used for inoculation of animals ineach group). Consistent with the level of viremia, the neutralizingtiters in monkeys immunized with the PMS virus (without mutation) werehigher than the other 2 groups (p=0.0002). The sera of Group 1 monkeys(immunized with a DEN1 chimera with 2 mutations on the envelopeproteins, prM and E) revealed the lowest neutralizing titers (Table 9),indicating that the M39 mutation may have further attenuated the virus(p=0.0045). These experiments demonstrated that there might be a directcorrelation for ChimeriVax™-DEN viruses between 1) the magnitude ofviremia and the level of neutralizing antibodies in monkeys, and 2)neurovirulence of chimera for mouse and viremia/immunogenicity inmonkeys (clone E was attenuated for 4 days old mice and induced a lowerlevel of viremia and neutralizing antibodies than the PMS virus, whichwas neurovirulent for mice of similar age).

In summary, the mutation at E204 residue of ChimeriVax™-DEN1 controlsthe replication of the DEN1 chimera in vertebrate hosts, as shown byviremia and neutralizing responses. Mutation of this residue, which isconserved in all dengue serotypes (Table 10), can thus be used in theconstruction of chimeras with desired phenotypes appropriate for humandengue vaccine.

TABLE 1 Comparison of the amino acid differences in the E protein ofChimeriVax ™-JE FRhL₃ and ChimeriVax ™-JE FRhL₅ virus with publishedsequences of JE SA14-14-2 vaccine, wild-type JE strains, parental SA14,and Nakayama virus. ChimeriVax ™-JE FRhL₃ and FRhL₅ viruses weresequenced across their entire genomes and the mutation at E279 was theonly difference found. Virus E107 E138 E176 E177 E227 E244 E264 E279E315 E439 ChimeriVax ™-JE FRhL₃ E279 Met F K V A S G H M V RChimeriVax ™-JE FRhL₅ E279 Lys F K V A S G H K V R JE SA14-14-2 PDK¹ F KV T S G Q M V R JE SA14-14-2 PHK² F K V A S G H M V R JE SA14^(1,3) L EI T S G Q K A K JE Nakayama⁴ L E I T P E Q K A K ¹Nitayaphan S. et al.1990. Virology 177: 541-552 ²Ni H. et al. 1994. J. Gen. Virol. 75:1505-1510; PDK = primary dog kidney ³Aihara S. et al. 1991. Virus Genes5: 95-109; PHK = primary hamster kidney ⁴McAda P. et al. 1987. Virology158: 348-360

TABLE 2 Neurovirulence for suckling mice of ChimeriVax ™-JE viruses withand without a mutation at E279 and YF 17D vaccine Mouse Virus, passageIntracerebral Average LD₅₀ age and E279 amino dose Mortality Survival(Log₁₀ Experiment (days) acid (Log₁₀ PFU) (%) Time (Days) PFU) 1 8.6YF-VAX ® 1.15 10/10 (100) 8.4 0.11 0.15 5/10 (50) 10 0-0.85 1/10 (10) 14ChimeriVax ™- 2.60 1/10 (10) 15 >2.6 JE, FRhL_(3,) 1.6 1/10 (10) 13 E279Met 0.6 0/10 (0)  N/A −0.45 0/10 (0)  N/A ChimeriVax ™- 3.0 10/10 (100)10.3 1.64 JE, FRhL_(5,) 2.0 8/10 (80) 11.25 E279 Lys 1.0 2/10 (20) 14.50 2/10 (20) 16 2 4 YF-VAX ® 0.95 11/11 (100) 8.4 −0.3 −0.05 9/11 (82)8.8 −1.05 2/12 (17) 10 ChimeriVax ™- 2.69 7/12 (58) 10.6 2.5 JE,FRhL_(3,) 1.69 4/12 (33) 11.5 E279 Met 0.69 0/12 (0)  NA ChimeriVax ™-2.88 10/12 (83)  9.3 1.45 JE, FRhL_(5,) 1.88 11/12 (92)  10.3 E279 Lys0.88 4/12 (33) 12.2 −0.11 2/12 (17) 14 −1.11 0/12 (0)  NA YF/JE₂₇₉ site-3.55 12/12 (100) 9.4 1.15 specific 2.55 11/12 (92)  10.1 revertant, 1.5511/12 (92)  10.2 E279 Lys 0.55 3/12 (25) 10.7 −0.44 2/12 (17) 14

TABLE 3 Neuropathological evaluation, monkeys inoculated IC withChimeriVax ™-JE FRhL₃, FRhL₅ or yellow fever 17D (YF-VAX ®) andnecropsied on day 30 post- inoculation. Clinical Individual and groupmean Dose¹ score² histopathological score log₁₀ Maximum Target + PFU/score/Mean Target Discriminator Discriminator Test virus Monkey Sex 0.25mL daily score area³ areas⁴ areas YF-VAX ® RT702M M 4.05 1/0 2.00 0.511.26 Connaught RT758M M 4.28 1/0 0.25 0.01 0.13 Lot # 0986400 RT653M M4.07 1/0 2.00 0.39 1.20 RT776M M 4.25 3/1 2.00 1.29 1.65 RT621M M 4.343/2 1.00 0.46 0.73 RAH80F F 4.14 3/1 1.50 0.71 1.10 RAL02F F 4.13 1/12.00 0.80 1.40 RT698F F 3.78 3/1 1.50 0.64 1.07 RAI12F F 4.11 1/1 2.001.45 1.73 RP942F F 4.05 1/0 2.00 0.81 1.41 Mean 4.12 1 1.63 0.71 1.17 SD0.16 1 0.59 0.42 0.47 ChimeriVax ™- RT452M M 3.55 1/0 0.50 0.08 0.29 JE,FRhL₃ RR257M M 3.52 1/0 1.00 0.14 0.57 Lot# I031299A RT834M M 3.71 1/00.50 0.38 0.44 RT620M M 3.71 1/0 1.00 0.14 0.57 RT288M M 3.76 1/0 0.500.19 0.35 RAJ98F F 3.79 1/1 0.00 0.11 0.05 RAR08F F 3.52 1/0 0.00 0.130.07 RV481F F 3.52 1/0 0.00 0.06 0.03 RT841F F 3.71 1/0 0.50 0.05 0.28RT392F F 3.76 1/0 0.50 0.07 0.29 Mean 3.66 0 0.45 0.14 0.29 SD 0.11 00.37 0.10 0.20 P-value (t Test⁵) vs. YF-VAX ® 0.037/0.025 0.000080.00191 0.00014 ChimeriVax ™- RT628M M 4.20 1/0 0.50 0.57 0.54 JE, FRhL₅RT678M M 4.19 1/0 1.00 0.12 0.60 Lot # 99B01 RT581M M 4.17 1/0 1.00 0.460.73 RR726M M 4.32 1/0 1.00 0.66 0.83 RR725M M ND⁶ 1/0 1.00 0.33 0.67RAJ55F F 4.27 0/0 1.00 0.14 0.57 RT769F F 4.44 1/0 1.00 0.58 0.79 RAK22FF 4.24 1/0 0.00 0.12 0.06 RT207F F 4.49 1/1 1.00 0.22 0.61 RT490F F 4.341/0 0.00 0.04 0.02 Mean 4.30 0 0.75 0.32 0.54 SD 0.11 0 0.42 0.23 0.28P-value (t Test) vs. YF-VAX ® 0.024/0.025 0.00154 0.02436 0.00248P-value (t Test) vs. ChimeriVax ™-JE FRhL₃ 0.343/1.00  0.10942 0.032230.03656 ¹Back-titration ²Clinical score: 0 = no signs; 1 = rough coat,not eating; 2 = high pitched voice, inactive, slow moving; 3 = tremor,incoordination, shaky movements, limb weakness; 4 = inability to stand,paralysis, moribund, or dead. The maximum score on any day and the meanscore over the 30-day observation period are shown. ³Substantia nigra⁴Corpus striatum and thalamus, right and left side (N. caudatus, globuspallidus, putamen, N. ant./lat. thalami, N. lat. thalami; cervical andlumbar enlargements of the spinal cord (6 levels) ⁵Student's t test,two-sided, heteroscedastic, comparing YF-VAX ® and ChimeriVax ™-JEviruses. ⁶Not done

TABLE 4 Viremia, rhesus monkeys inoculated IC with YF-VAX ® orChimeriVax ™-JE FRHL₃ and FRHL₅ viruses (for dose inoculated, see Table3) Serum Virus Titer (Log₁₀ PFU/mL), Day Animal 1 2 3 4 5 6 7 8 9YF-VAX ® Control RT702M —¹ — 1.6 3.0 — — — — — RAH80F — — — 3.3 2.5 — —— — RT758M — — 2.1 3.2 2.8 — — — — RAL02F — — — 1.3 — — — — — RT653M — —— 2.7 — — — — — RT698F — 1.0 2.3 3.7 2.5 — 1.0 — — RT776M — — — — — — —— — RAI12F — — — 2.0 2.5 2.5 2.0 — — RT621M — 1.0 2.0 3.3 2.0 — — — —RP942F — 1.0 2.6 3.6 2.0 — — — — Mean Titer² 0.8 1.4 2.7 1.7 0.9 0.9 SD0.1 0.8 1.0 0.9 0.6 0.4 ChimeriVax ™-JE FRHL₃ E279 Met RAJ98F — — 1.91.3 — — — — — RT452M — 1.3 2.1 1.6 — — — — — RAR08F — — 1.3 2.2 2.2 1.8— — — RR257M — — 1.9 2.2 1.8 — — — — RV481F — — 2.1 1.8 1.5 — — — —RT834M — — 2.5 1.3 — — — — — RT841F — — 2.4 1.7 — — — — — RT620M — — 1.61.0 — — — — — RT392F — — — — — — — — — RT288M — — — — — — — — — MeanTiter 0.8 1.7 1.5 1.0 0.8 SD 0.2 0.6 0.5 0.6 0.3 P-value³  0.696  0.386 0.003  0.065  0.745 ChimeriVax ™-JE FRhL₅ E279 Lys RT628M — — — — — — —— — RAJ55F — — — — — — — — — RT678M — — — — — — — — — RT769F — — — 2.0 —— — — — RT581M — — — — — — — — — RAK22F — — — — — — 1.8 — — RR726M — — —— — — — — — RT207F — — — — — — — — — RR725M — — — — — — — — — RT490F — —— — — — — — — Mean Titer 0.7 0.7 0.8 0.7 0.7 0.8 SD 0.0 0.0 0.4 0.0 0.00.4 P-value⁴  0.331  <0.000  0.010  0.076 1.0 1.0 ¹— = No detectableviremia; in most tests neat serum was tested, the cutoff being 1.0 log₁₀PFU/mL); in some cases, neat serum was toxic to cells, and serum diluted1:2 or 1:5 was used (cut-off 1.3 or 1.7 log₁₀ PFU/mL). ²For the purposeof calculating mean titers and standard deviations, 0.7 was used inplace of <1.0, 1.0 was used in place of <1.3, and 1.4 was used in placeof <1.7. ³Comparison with YF-VAX ® by t-test, 2-tailed ⁴Comparison withChimeriVax ™JE FRhL₃ by t-test, 2-tailed

TABLE 5 Nucleotide and amino acid sequences of uncloned and variousclones of ChimeriVax- DEN1 viruses and their in vitro (Vero passages)genetic stabilities. Nt. change/ AA change/ Virus Passage Gene Nt.No^(a) heterogeneity heterogeneity AA No^(b) Comments Uncloned P2 — — —— — No mutations Uncloned P5 E 1590 A/G K/R 204 Nucleotide heterogeneityE 1730 G/T V/F 251 Nucleotide heterogeneity E 1912 G/t E/D 311 Barelydetectable mutant E 2282 C/a L/I 435 Undetectable mutants in somesamples Uncloned P15 E 1590 A to G K to R 204 NS2B 4248 G to T G to VNS4A 6888 C/T A/V Nucleotide heterogeneity NS4A 7237 A/G I/M Nucleotideheterogeneity Uncloned P15 E 1590 A to G K to R 204 REPEAT E 1730 G/TV/F 251 Nucleotide heterogeneity from P2 NS4A 7237 A/G I/M 263Nucleotide heterogeneity NS4B 7466 C/t P/S  52 Barely detectable mutantClone A P3, P7 E 1730 G to T V to F 251 Domain II j strand, no functionassigned E 2282 C to A L to I 435 Before anchor; L and I in D2 and YFrespectively. (a gap left, nt 7080-7220) Clone B P3, P7, P10 E 1730 G toT V to F 251 Clone C P3, P6 E 1912 G to T E to D 311 Domain III, astrand, no function assigned. Clone D P3, P6 E 1730 G to T V to F 251Clone E P3, P6 E 1590 A to G K to R 204 Domain II, f-g loop of, nofunction ass. Clone F P3 M  788 C to T — — E 1590 A to G K to R 204Clone G P3 E 1730 G to T V to F 251 Clone H P3 E 1912 G to T E to D 311E 2030 G to T V to L 351 Domain III, d strand (L in D2 and D3; I in D4)Clone I P3 E 1590 A to G K to R 204 Clone J P3, P6, P7, — — — — — (J-2)P10 Cline J P8 E 1590 A to G (a/G) K to R 204 Some parent (a) nucleotidestill (J-2) (cGMP MS) present P10 from E 1590 A to G K to R 204 (cGMPMS) Clone J P10 REPEAT E 1590 A to G K to R 204 (J-2) from P7 Clone JP20 From E 1590 A to G K to R 204 (J-2) P10 repeat NS4A 6966 G/T S/I 171NS4A 7190 G/a V/I 246 ^(a)From the beginning of the genome. ^(b)From theN-terminus of indicated protein; numbering according to Rice et al.,Science 229: 726-733, 1985. Clones with 204 mutations are shown in boldletters.

TABLE 6 Neurovirulence of different clones of chimeric DEN1 viruses in4-day old suckling mice. ChimeriVax- Dose No. dead/total AST DEN1Mutation Dilution (BT) (% dead) Days Uncloned None Neat 5.0 11/11 (100)9.1 (P2) 1:10 4.1 11/11 (100) 10.2 Clone B E251 Neat 5.8 10/11 (91)  9.8(P7) V to F 1:10 5.0 11/11 (100) 10.2 Clone C E311 Neat 5.8 11/11 (100)8.5 (P6) E to D 1:10 4.9 11/11 (100) 9.5 Clone E E204 Neat 5.9 3/11 (27)13 (P6) K to R 1:10 4.8 1/11 (9)  14  1:100 4.0 1/11 (9)  15 Clone JNone Neat 3.6 11/11 (100) 10.8 (P3) 1:10 3.0 11/11 (100) 11.3  1:100 1.89/11 (82) 11.3 YF-VAX ® NA 1:20 2.5 12/12 (100) 8.3

TABLE 7 Immunogenicity Study in Rhesus Monkeys, ChimeriVax ™-DEN1viruses, Sierra Biomedical NON-GLP Study Dose Group* Virus Mutation (0.5ml) 1 ChimeriVax-DEN-1 M39 and 5 logs Uncloned, P4, 1999** E204 2ChimeriVax-DEN-1, E204 5 logs P6, clone E 3 ChimeriVax-DEN-1, None 5logs PMS (P7), clone J *Four monkeys (2M/2F) per group. **Guirakhoo etal 2001

TABLE 8 Viremia in monkeys immunized with 5 log₁₀ PFU (S.C.) ofdifferent clones of ChimeriVax-DEN1 viruses Viremia (log₁₀ PFU/ml) byVirus post-immunization day: Monkey (Mutation) 2* 3 4 5 6 7 8 9 10 11R18265M ChimeriVax ™- —** — — — — — — — — — R175110F DEN1, — — — 1.7 — —— — — — R17572M 99, P4, 1.3 1.0 — 1.0 — — — — — — R171114F uncloned — —— — — — — — — — (M39, E204) R182103M ChimeriVax ™- — — — — — — — — — —R17098F DEN1, — 1.7 — — — — — — — — R18261M P6, clone 1.7 2.5 1.3 2.0 —R175118F E, (E204) — — 1.0 — — — — — — — R182104M ChimeriVax ™- 1.0 1.91.7 1.7 1.8 1.7 1.0 1.0 1.7 — R175108F DEN1, — 1.7 2.8 2.2 1.0 2.0 1.72.0 2.2 1.7 R182111M P7, clone 2.3 3.0 3.3 2.8 1.7 1.7 — — — — R175104FJ, PMS — 2.4 1.3 2.0 2.3 1.7 1.7 2.2 3.0 3.1 (None) *Monkeys wereimmunized on Day 1. **<1.0 log10 PFU/ml

TABLE 9 Viremia and neutralizing antibody titers (50%) in monkeysimmunized with 5 log₁₀ PFU (S.C.) of different clones of ChimeriVax-DEN1viruses No. Plaque viremic/no. Mean Neut. Ab size Monkey Mutation tested(%) Peak titer Duration titer (mm) R18265M YF-DEN1, 99, 2/4 (50) 1.5 1.5640 2-4 R175110F P4, uncloned 640 R17572M (M39, E204) 320 R171114F 640R182103M YF-DEN1, 01, 3/4 (75) 1.7 2 5120 2-4 R17098F P6, clone E 2560R18261M (E204) 2560 R175118F 5120 R182104M YF-DEN1, 01, 4/4 (100) 2.58.5 5120 1 R175108F P7, clone J, 10240 R182111M PMS 10240 R175104F(None) 10240

TABLE 10 Position of 204 residues in ChimeriVax ™-DEN1-4 E proteinsAmino Acid residues, E protein ChimeriVax- 200 201 202 203 204 205 206207 208 DEN1 T M K E K S W L V DEN2 Q M E N K A W L V DEN3 T M K N K A WM V DEN4 K M K K K T W L V

What is claimed is:
 1. A method of reducing the viscerotropism and/orneurovirulence of a flavivirus which is a chimeric flavivirus comprisingsequences encoding capsid and non-structural proteins of a firstflavivirus and pre-membrane and envelope proteins of a dengue virusselected from a dengue-1 virus, a dengue-2 virus, a dengue-3 virus, anda dengue-4 virus, the method comprising substituting lysine at envelopeprotein position 202 (dengue-3) or 204 (dengue-1, dengue-2, or dengue-4)with arginine.
 2. The method of claim 1, wherein said sequences encodingsaid dengue pre-membrane and envelope proteins encode pre-membrane andenvelope proteins of a dengue-1 virus.
 3. The method of claim 1, whereinsaid sequences encoding said dengue pre-membrane and envelope proteinsencode pre-membrane and envelope proteins of a dengue-2 virus.
 4. Themethod of claim 1, wherein said sequences encoding said denguepre-membrane and envelope proteins encode pre-membrane and envelopeproteins of a dengue-3 virus.
 5. The method of claim 1, wherein saidsequences encoding said dengue pre-membrane and envelope proteins encodepre-membrane and envelope proteins of a dengue-4 virus.
 6. The method ofclaim 1, wherein said first flavivirus is a yellow fever virus.
 7. Themethod of claim 6, wherein said substitution reduces the viscerotropismof said chimeric flavivirus.
 8. A method of inducing an immune responseto a flavivirus in a subject, said method comprising administering tosaid patient a flavivirus which is a chimeric flavivirus comprisingsequences encoding capsid and non-structural proteins of a firstflavivirus and pre-membrane and envelope proteins of a dengue virusselected from a dengue-1 virus, a dengue-2 virus, a dengue-3 virus and adengue-4 virus, wherein the dengue virus envelope protein has asubstitution of the lysine at amino acid position 202 (dengue-3) or 204(dengue-1, dengue-2, or dengue-4) with arginine and wherein said methodresults in decreased viscerotropism and/or neurovirulence in saidsubject relative to a corresponding flavivirus lacking the substitution.9. The method of claim 8, wherein said sequences encoding said denguepre-membrane and envelope proteins encode pre-membrane and envelopeproteins of a dengue-1 virus.
 10. The method of claim 8, wherein saidsequences encoding said dengue pre-membrane and envelope proteins encodepre-membrane and envelope proteins of a dengue-2 virus.
 11. The methodof claim 8, wherein said sequences encoding said dengue pre-membrane andenvelope proteins encode pre-membrane and envelope proteins of adengue-3 virus.
 12. The method of claim 8, wherein said sequencesencoding said dengue pre-membrane and envelope proteins encodepre-membrane and envelope proteins of a dengue-4 virus.
 13. The methodof claim 8, wherein said first flavivirus is a yellow fever virus. 14.The method of claim 13, wherein said substitution reduces theviscerotropism of said chimeric flavivirus.
 15. The method of claim 8,further comprising administering to said subject three additionalchimeric flaviviruses that each comprise sequences encoding capsid andnon-structural proteins of said first flavivirus and pre-membrane andenvelope proteins of one of dengue-1, dengue-2, dengue-3, and dengue-4viruses, wherein the dengue virus sequences of the three additionalchimeric flaviviruses are of serotypes that are different from oneanother and different from that administered in the method of claim 8,and optionally the dengue virus envelope protein of one or more of thethree additional chimeras has a substitution of the lysine at amino acidposition 202 (dengue-3) or 204 (dengue-1, dengue-2, or dengue-4) witharginine.
 16. A method of reducing the viscerotropism and/orneurovirulence of a flavivirus which is a dengue virus selected from adengue-1 virus, a dengue-2 virus, a dengue-3 virus, and a dengue-4virus, the method comprising substituting lysine at envelope proteinposition 202 (dengue-3) or 204 (dengue-1, dengue-2, or dengue-4) witharginine, wherein when viscerotropism and/or neurovirulence of saiddengue-3 virus is reduced, said substitution of lysine at envelopeprotein position 202 with arginine is the only mutation in said dengue-3virus.
 17. The method of claim 16, wherein said dengue virus is adengue-1 virus.
 18. The method of claim 16, wherein said dengue virus isa dengue-2 virus.
 19. The method of claim 16, wherein said dengue virusis a dengue-3 virus.
 20. The method of claim 16, wherein said denguevirus is a dengue-4 virus.
 21. The method of claim 16, wherein saidsubstitution reduces the viscerotropism of said flavivirus.
 22. A methodof inducing an immune response to a flavivirus in a subject, said methodcomprising administering to said patient a flavivirus which is a denguevirus selected from a dengue-1 virus, a dengue-2 virus, a dengue-3virus, and a dengue-4 virus, wherein the dengue virus envelope proteinhas a substitution of the lysine at amino acid position 202 (dengue-3)or 204 (dengue-1, dengue-2 or dengue-4) with arginine and wherein saidmethod results in decreased viscerotropism and/or neurovirulence in saidsubject relative to a corresponding flavivirus lacking the substitution.23. The method of claim 22, wherein said dengue virus is a dengue-1virus.
 24. The method of claim 22, wherein said dengue virus is adengue-2 virus.
 25. The method of claim 22, wherein said dengue virus isa dengue-3 virus.
 26. The method of claim 22, wherein said dengue virusis a dengue-4 virus.
 27. The method of claim 22, wherein saidsubstitution reduces the viscerotropism of said flavivirus.